The narrow 2012 Grand National runner-up Sunnyhillboy will miss next Saturday's big race.
Trainer Jonjo O'Neill said the horse, beaten a nose by Neptune Collonges two years ago, was "lacking sparkle".
Sunnyhillboy had been well backed to as low as 16/1 amid speculation 18-time champion jockey AP McCoy might take the ride at Aintree on 5 April.
"He's been a super horse for us but he just didn't sparkle in his work [on Friday morning]," O'Neill said.
"Going for a race like that you have to be spot on and he just hasn't done what we'd have wanted in his last few bits of work.
"He's been great and we hope he'll be back next season."